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Oxidative stress after stroke is associated with the inflammatory system activation in the brain. The complement cascade, especially the degradation products of complement component 3, is a key inflammatory mediator of cerebral ischemia. We have shown that pro-inflammatory complement component 3 is increased by oxidative stress after ischemic stroke in mice using DNA array. In this study, we investigated whether up-regulation of complement component 3 is directly related to oxidative stress after transient focal cerebral ischemia in mice and oxygen-glucose deprivation in brain cells. Persistent up-regulation of complement component 3 expression was reduced in copper/zinc-superoxide dismutase transgenic mice, and manganese-superoxide dismutase knock-out mice showed highly increased complement component 3 levels after transient focal cerebral ischemia. Antioxidant N-tert-butyl-α-phenylnitrone treatment suppressed complement component 3 expression after transient focal cerebral ischemia. Accumulation of complement component 3 in neurons and microglia was decreased by N-tert-butyl-α-phenylnitrone, which reduced infarct volume and impaired neurological deficiency after cerebral ischemia and reperfusion in mice. Small interfering RNA specific for complement component 3 transfection showed a significant increase in brain cells viability after oxygen-glucose deprivation. Our study suggests that the neuroprotective effect of antioxidants through complement component 3 suppression is a new strategy for potential therapeutic approaches in stroke.
Stroke is the rapidly developing loss of brain function because of failure of cerebral blood flow (CBF). It is the third leading cause of death worldwide, and the mortality rate has not improved during the past two decades. Reperfusion after cerebral ischemia generates additional overproduction of free radicals, which leads to secondary brain injury (Taskapilioglu et al. 2009). The brain is a vulnerable target for reactive oxygen species (ROS)–induced damage for many reasons, such as high oxygen consumption, a low level of protective antioxidants, and a high concentration of peroxidizable lipids. Oxygen deprivation causes nerve cells in the affected area to die within minutes.
Complement is a host defense system that identifies pathogens and injured cells, recruits inflammatory cells, and induces cell lysis (del Zoppo 1999). The complement cascade has three different pathways: the classical pathway, the mannose-binding lectin pathway, and the alternative pathway. The initiators of the three pathways are different, but all three ultimately lead to activation of complement component 3 (C3). This is the crucial step for the biological activity of the complement system because C3-cleavage byproducts lead to further downstream activation, which generates responses such as anaphylaxis, chemotaxis, and phagocytosis (Daha 2010). It is also reported that there is a fourth extrinsic protease pathway, which generates C5a in the absence of C3 (Huber-Lang et al. 2006).
It was once thought that the blood–brain barrier blocks the brain from the immune system. However, astrocytes, microglia, and neurons produce complement to maintain immunosurveillance in the brain (Gasque et al. 2000). We know that ischemic stroke increases complement levels and results in neurological disability (Cojocaru et al. 2008). C3 deficiency is associated with a better outcome after acute stroke (Cervera et al. 2010). C3a and C5a are anaphylatoxins that are bioactive fragments of C3 and C5. These anaphylatoxins induce release of various mediators from mast cells and phagocytes that amplify inflammatory responses. Inhibition of the complement cascade produces a better outcome in heart, liver, and brain after ischemic damage (Mocco et al. 2006; Ducruet et al. 2009). Although numerous approaches have been tried to reduce ischemic damage, including inhibition of C1, C3, C5, and membrane attack complex, and using cobra venom factor for complement depletion, none has reached the market (Arumugam et al. 2009; Qu et al. 2009). Although many studies have been done to understand the relationship between complement components and stroke, the role of the complement cascade in cerebral ischemia is still not fully understood.
Here, we studied the relationship between C3 up-regulation and oxidative stress after transient focal cerebral ischemia (tFCI). To examine how complement components depend on oxidative stress after ischemic stroke, we used copper/zinc-superoxide dismutase transgenic (SOD1 Tg) mice and manganese-superoxide dismutase knock-out (SOD2 KO) mice. There were no significant differences in glutathione and ascorbate levels between SOD1 Tg and wild-type (WT) mice after focal cerebral ischemia (Kinouchi et al. 1991). As glutathione and catalase system are intact in SOD1 Tg and SOD2 KO mice, the total ROS levels were increased not because the compensatory reaction but because oxidative stress from ischemic damage. We pharmacologically studied the inhibitory role of the antioxidant N-tert-butyl-α-phenylnitrone (PBN) in complement activation after tFCI in mice. We also specifically knocked-down C3 level to figure out specific role of C3 after ischemic injury, and measured C3 plasma level. The goal of this study was to elucidate the interactions between complement cascade activation and oxidative stress after cerebral ischemia.
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ROS are a fundamental mechanism of brain damage and the excessive production of ROS is associated with oxidative stress (Allen and Bayraktutan 2009). Growing evidence suggests that stroke, the third leading cause of death in the world, is related to immunological inflammation. The complement system is an integral part of the immune defense mechanism and is also a primary mediator of the inflammatory process (Frank and Fries 1991). The complement cascade is activated in the brain after cerebral I/R, and there is significant evidence that complement components are deposited after cerebral ischemic injury (Cowell et al. 2003). Previous studies have shown that complement levels were increased after ischemic stroke and that complement pathway inhibition by C1q and C3 inhibition and mannose-binding lectin deficiency lead to a better neurological outcome (Cojocaru et al. 2008; Huang et al. 2008; Cervera et al. 2010). A recent study identified C3 as the most influential mediator of ischemic injury in the complement cascade (Mocco et al. 2006). C3 activation was increased at 72 h, and complement depletion significantly reduced brain edema 72 h after intracerebral hemorrhage (Vakeva et al. 1994; Xi et al. 2001). Consistent with earlier reports, we found that C3 levels were increased after tFCI up to 7 days in mice. However, conflicting views have emerged about the role of complement components. Complement has a role in CNS regeneration, and C3a and C5a are crucial for hepatocyte proliferation and liver regeneration in mammalian systems (Mastellos et al. 2001; Strey et al. 2003; Daveau et al. 2004; Rahpeymai et al. 2006). However, excessive complement activation leads to severe damage after stroke, and reoxygenation of anoxic endothelial cells has also been shown to increase complement activation and deposition (Väkevä and Meri 1998). This implies that complement activation has both protective and deleterious effects, so it should be modulated rather than blunted. In this study, we used SOD2 KO and SOD1 Tg mice to find the close relationship between C3 activation and excessive superoxide radicals. Our results showed that SOD2 KO mice with higher ROS levels showed higher C3 level and SOD1 Tg mice brains had decreased C3 level after I/R. The natural free-radical scavenger, SOD, reduced ischemic brain injury and simultaneously reduced C3 level. These data imply that complement activation after cerebral I/R is directly related to oxidative stress.
Previous studies have shown the relationship between the complement cascade and antioxidants in heart I/R models. The mechanisms of cardioprotection after ischemia appear to be antioxidant activity and direct scavenging of superoxide anions, as well as a reduction in the levels of the C-reactive protein and membrane attack complex in infarcted tissue (Lockwood and Gross 2005). In a gastrointestinal I/R model, anti-complement treatment preserved SOD1 activity and decreased oxidative stress (Montalto et al. 2003). However, how complement components and antioxidants are related in brain I/R injury has not yet been discovered. Thus, we studied the effect of an antioxidant along with the complement cascade activation in a brain ischemia model to investigate whether oxidative stress is directly related to complement component levels after tFCI.
Over the last few years, free-radical scavengers, such as ebselen and the radical-trapping agent NXY-059, have gained considerable attention in the field of stroke research (Allen and Bayraktutan 2009). However, in a clinical trial, the use of ebselen had a disappointing outcome, compared with a placebo, at 3 months. Another phase III study was slated to begin in 2001, but no report has appeared (Yamaguchi et al. 1998; Ginsberg 2008). NXY-059 scores mirrored those of the placebo group (Ginsberg 2008). There have been no commonly used antioxidant drugs for stroke treatment yet. In this study, we used the antioxidant PBN to examine whether it can reduce C3 activation for neuroprotection after cerebral ischemia. PBN is a spin-trapping agent that shows neuroprotection against brain injury and it was previously reported that PBN did not change CBF as we confirmed (Miyajima and Kotake 1995; Liu et al. 2003). Earlier studies revealed that PBN shows neuroprotective effects by improving mitochondrial function and recovery of the cerebral energy state after tFCI, and by attenuating lactate formation, in addition to free-radical scavenging (Folbergrová et al. 1995; Kuroda et al. 1996; Lewén and Hillered 1998). PBN also activates extracellular signal-regulated kinase, suppresses stress-activated protein kinase/c-Jun N-terminal kinase and p38 activation, and increases expression of heat-shock proteins 27 and 70 (Tsuji et al. 2000). The anti-inflammatory mechanism of PBN in the immune system after cerebral ischemia has not been examined nor extensively researched. Our results showed that PBN reduced C3 mRNA level which was increased by tFCI, and nuclear accumulation of C3-related proteins, which suggests that PBN regulates the C3 level during cerebral I/R in mice. The neurological deficit gradually recovered as previously reported, but PBN significantly improved behavioral recovery compared to vehicle (Yang et al. 2009). PBN also reduced infarct volume.
Our study also found that the anaphylatoxins C3a and C5a were increased after tFCI, but were reduced by PBN administration. These results correspond with previously reported studies that C3a and C5a also play a deleterious role in ischemic stroke (Ducruet et al. 2012; Pavlovski et al. 2012). Many biological activities are associated with complement activation, including formation of anaphylatoxins such as C3a and C5a (Chakraborti et al. 2000). Other downstream factors of C3, such as C3b, C3d, C5 and membrane attack complex, also induce neuronal death after ischemic stroke (Ducruet et al. 2012; Pavlovski et al. 2012; Arumugam et al. 2007). Administration of C3d reduces primary neural progenitor cells proliferation, and antagonism of the C3a receptor promotes proliferation of migrating neuroblasts after stroke (Ducruet et al. 2012). Endogenously generated C5a also exacerbated neuronal apoptosis under ischemic condition (Pavlovski et al. 2012).
It has been shown that brain cells themselves were able to produce complement components (Lévi-Strauss and Mallat 1987). Among the numerous brain cells, microglia and neurons produce almost all complement proteins (Morgan and Gasque 1996; Gasque et al. 2000). Our double immunofluorescent staining results showed that neurons and microglia changed their morphology to activated form after tFCI and that C3 was co-localized in them. Previous studies reported that complement component binds to nucleic acid in lupus and inner-nuclear layers of retina (Papp et al. 2010; Amadi-Obi et al. 2012). On the basis of these, we hypothesize that normally, C3 is mainly located in the cytosol and is translocated to the nucleus when the complement cascade is activated by oxidative stress and has a deleterious role. However, further study is needed to figure out the specific role and mechanism of activated C3 in nuclear. In mouse brain primary cortical neurons, inhibition of C3 by treatment with C3 siRNA decreased cell cytotoxicity after OGD. Furthermore, intracellular ROS levels, which were highly up-regulated by OGD, were significantly decreased by C3 siRNA-transfection. These results indicate that excessive C3 produced by ROS has a deleterious role in neurons after I/R injury in mice.
Despite these findings, the mechanisms governing the relationship between oxidative stress and C3 activation have been unknown. To further investigate the redox-sensitive mechanisms of C3 activation, promoter research is much needed. The C3 promoter has many oxidative stress– and inflammation-related factors such as activator protein-1, interleukin-1β, p38, and CCAAT/enhancer-binding protein delta (C/EBPδ) (Juan et al. 1993; Schaefer et al. 2005; Maranto et al. 2008). Interleukin-1β regulates the C3 gene in astrocytes and C/EBPδ is a pivotal transcription factor involved in brain inflammation (Cardinaux et al. 2000; Maranto et al. 2008). Also, activator protein-1 plays a role in C3a receptor expression (Schaefer et al. 2005). In this study, we transiently transfected C3 plasmid which contains C3 promoter region to mouse primary cortical neurons, subjected 3 h of OGD and reperfused 6 h, 24 h, 48 h, and 72 h. The promoter was highly activated after OGD up to 72 h. It has been known that oxidative stress peaks at a much earlier time point, but an inflammatory reaction occurs because of secondary ROS (Spychalowicz et al. 2012; Yang et al. 2012). Thus, C3 might elevate because of inflammation after stroke. As C3 promoter is activated, interleukin-1 and interleukin-6 levels are also elevated and these lead to secondary ROS production (Won and Baumann 1990; Vik et al. 1991; Jendrysik et al. 2011). Hence, our results suggest that down-regulation of C3 also lowers ROS level. To clarify the relationship between C3 and oxidative stress, further research of C3 promoter and its transcriptional factors are needed.
As the peripheral and the brain immune system are not separated, but connected; the immune response after tFCI is systemic including the brain (Yong and Rivest 2009; Crehan et al. 2012). Thus, cerebral C3 might come from both CNS cells and peripheral cells. Another possibility that brain and peripheral system are separated and activates C3 independently has been suggested (van Beek et al. 2003). The specific mechanism between the CNS and the peripheral system needs to be further studied. Growing evidence suggests that inflammatory markers can predict stroke since inflammation plays an important role in pathophysiology of brain ischemia (Vibo et al. 2007; Whiteley et al. 2012). Developing blood markers to diagnose and prevent stroke has been done in many research groups. It has been previously reported that glial fibrillary acidic protein, tau, lipoprotein-associated phospholipase A2, and granulocyte colony-stimulating factor levels in blood were increased in stroke patients (Nylen et al. 2007; Hu et al. 2012; Schiff et al. 2012; Tsai et al. 2012; Yu et al. 2012). Also, plasma C3 levels were elevated in cryptogenic and large-vessel disease stroke, and high plasma C3 levels were related with unfavorable outcomes only in large-vessel disease stroke (Stokowska et al. 2011). Our results showed that C3 plasma levels were increased after tFCI and were down-regulated by antioxidant PBN administration. C3 plasma levels also showed co-relation with behavioral change. Thus, plasma C3 levels measurement has possibility to diagnose and prevent stroke in a clinical setting.
The complement cascade is activated after cerebral ischemia and plays a deleterious role, and oxidative stress is closely associated with C3 activation. Mice with lower ROS levels showed a better neurological outcome with lower C3 levels after tFCI. In this study, we identified not only a causal relationship between ROS and C3 activation in stroke but also a new anti-inflammatory effect of PBN through suppression of C3 activation in mice. An antioxidant that is able to inhibit complement C3 activation may offer the new strategy for therapeutic approaches in stroke patients.